This project is centered on the mechanisms of calcium-triggered exocytosis, the ubiquitous eukaryotic process by which vesicles fuse to the plasma membrane and release their contents. SNAREs such as VAMP, SNAP-25 and syntaxin are essential for intracellular trafficking, but what are their exact molecular roles and how are their interactions with other proteins manifest? Capitalizing on the differential sensitivity of SNAREs to exogenous proteases, we quantified the selective removal of identified SNAREs from native secretory vesicles without loss of fusion competence. Using previously established fusion assays and a high sensitivity immunoblotting protocol, we analyzed the relationship between these SNARE proteins and Ca2+-triggered membrane fusion. Neither the extent of fusion nor the number of intermembrane fusion complexes per vesicle were correlated with the measured density of identified egg cortical vesicle (CV) SNAREs. Without syntaxin, CVs remained fusion competent. Surprisingly, for one (but not another) protease the Ca2+ dependence of fusion was correlated with CV SNARE density, suggesting a native protein complex that associates with SNAREs, the architecture of which ensures high Ca2+ sensitivity. As SNAREs may function during CV docking in vivo, and as further proteolysis after SNARE removal eventually ablates fusion, we hypothesize that the triggered steps of regulated fusion (Ca2+ sensitivity and the catalysis and execution of fusion) require additional proteins that function downstream of SNAREs. We have also proposed a new model for the cycle of exocytosis and endocytosis that can explain the regulation of transient fusion during exocytosis. With non-structural curving proteins such as epsin, which can change the spontaneous curvature (Js) of a membrane by insertion, it is possible to imagine a scenario by which cells regulate the activity of these curving proteins to effect local budding and flattening of membranes in endocytosis and exocytosis. For example, PIP2 accumulation may lead to epsin binding, thus increasing Js and the free energy of the patch of membrane (E). The lipids respond by budding into a vesicle with the same actual curvature (J), relieving the bending stress. Clathrin, which binds to epsin, can then polymerize into a cage around the vesicle, perhaps displacing epsin (which may remain at the neck to facilitate pinching off). When the vesicle is completely detached, it uncoats. Once epsin and clathrin are gone, the spontaneous curvature of the bilayer returns to 0, but as the membrane is actually curved, stress returns and the vesicle becomes metastable (E=500 kBT) This provides a stressed vesicle having a driving force for flattening out during exocytosis. For any vesicle about to fuse, by regulating the spontaneous curvature of the membrane, through proteins such as epsin, cells can regulate the degree to which fusion pores open and remain small, or repeatedly open and close "kiss and run" rather than flattening out in complete fusion.